Regulation of ubiquitin-dependent cargo sorting by
multiple endocytic adaptors at the plasma membrane
Jonathan R. Mayersa, Lei Wanga, Jhuma Pramanika, Adam Johnsona, Ali Sarkeshikb, Yueju Wangb,
Witchuda Saengsawangc, John R. Yates IIIb, and Anjon Audhyaa,1
Departments of aBiomolecular Chemistry and cNeuroscience, University of Wisconsin–Madison School of Medicine and Public Health, Madison, WI 53706; and
b
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037
Edited by Pietro De Camilli, Howard Hughes Medical Institute, Yale University, New Haven, CT, and approved June 4, 2013 (received for review
February 13, 2013)
clathrin
| multivesicular endosome
T
he directed movement of proteins and lipids between subcellular organelles is typically mediated by vesicular transport
carriers. A variety of coat protein complexes have been shown to
promote the membrane deformation steps that are necessary for
vesicle biogenesis (1, 2). In particular, the clathrin coat functions
at multiple intracellular locations to package a range of cargoes
for transport (3, 4). However, clathrin does not engage substrates
directly nor does it exhibit the ability to associate with membranes. Instead, recruitment of clathrin is mediated by specific
adaptor proteins found on different organelles, which interact
with cargoes and lipid bilayers (5). At the plasma membrane,
several endocytic adaptors coordinately regulate clathrin accumulation. The best characterized is adaptor protein complex 2
(AP-2), a heterotetrameric adaptor complex that can simultaneously bind to clathrin, sorting signals on cargoes and phosphatidylinositol 4,5-bisphosphate, a lipid specifically enriched on
the cell surface (6). Additionally, AP-2 associates directly with
other endocytic adaptors including the Eps15 homology (EH) domain containing proteins Eps15 and intersectin, members of the
muniscin family such as FCHO1, and the cargo-binding factors
Dab2 and Epsin (3, 7, 8). Thus, the AP-2 complex is a multifunctional scaffold that promotes the formation of cargoladen, clathrin-coated subdomains on the plasma membrane.
Despite its central role in clathrin-mediated endocytosis, the
AP-2 complex may not be the first to appear at all future sites of
budding. When overexpressed, the FCHO proteins, Eps15, and
intersectin may precede the arrival of AP-2 and clathrin on the
plasma membrane, although all of these factors exhibit highly
similar recruitment kinetics (7, 9, 10). Current models suggest
that the FCHO proteins initiate membrane bending and cargo
clustering via their FCH-BAR (F-BAR) and μ-homology domains,
www.pnas.org/cgi/doi/10.1073/pnas.1302918110
respectively (7, 8, 11). Simultaneously, Eps15 and intersectin recruit
downstream adaptors and accessory proteins, which collectively
drive maturation of the nascent endocytic pit (12). The early
recruitment of FCHO1/2, Eps15, and intersectin to the plasma
membrane suggest they play a role in the nucleation of clathrinmediated endocytosis (7, 13). However, this idea remains highly
controversial (7, 8, 14).
A large number of cell surface molecules undergo internalization
in a clathrin-dependent fashion. This process requires multiple endocytic adaptors to recognize largely distinct cargoes
in a manner that relies on short signal sequences or posttranslational modifications found within substrates (15). For
example, the addition of short ubiquitin chains to transmembrane
receptors plays a key role in their sorting from the plasma membrane to the lysosome for degradation (16). The adaptors Eps15
and epsin, which each harbor ubiquitin-binding domains, have
been shown to associate with ubiquitin-modified cargoes and direct them into the endolysosomal pathway (17). However, recent
studies suggest that the Eps15 ubiquitin interacting motifs may be
dispensable for receptor internalization (18). Moreover, yeast cells
lacking homologs of Eps15 and epsin continue to remove ubiquitin-modified receptors from the plasma membrane (19). These
findings suggest the existence of additional endocytic adaptors that
recognize ubiquitin-modified substrates at the cell surface.
Results
A Heterooligomeric Complex of FCHO-1, EHS-1, and ITSN-1 Functions
Specifically at the Plasma Membrane in Caenorhabditis elegans. To
address the function of endocytic adaptor proteins in cargo
sorting through the endolysosomal system, we used C. elegans as
a model system. In contrast to mammalian cells, which express
multiple isoforms of the FCHO proteins, Eps15, and intersectin,
the C. elegans genome encodes single homologs of each adaptor
(Fig. S1), which dramatically simplifies functional analysis. To
determine the subcellular distributions of these factors during
early stages of embryogenesis, we generated a set of affinitypurified antibodies (Fig. 1A), which were used in immunofluorescence studies. Consistent with previous data obtained from
cultured cells (7, 8, 14), we found that endogenous FCHO-1,
EHS-1, and ITSN-1 localized to the plasma membrane in
C. elegans embryos (Fig. 1B and Fig. S2 A and B). However, there
was a notable absence of specific EHS-1 and ITSN-1 staining on
intracellular membranes, as was reported for their mammalian
counterparts (20). In particular, we failed to observe colocalization of either protein with several, characterized endosomal
markers, including EEA-1 and RAB-5 (Fig. 1C). To further verify
Author contributions: J.R.M., L.W., and A.A. designed research; J.R.M., L.W., J.P., A.J., A.S.,
Y.W., W.S., and A.A. performed research; J.R.Y. and A.A. contributed new reagents/analytic tools; J.R.M., L.W., J.P., A.J., A.S., Y.W., and A.A. analyzed data; and J.R.M. and A.A.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1302918110/-/DCSupplemental.
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CELL BIOLOGY
Endocytic protein trafficking is directed by sorting signals on cargo
molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during
endocytosis remain poorly defined. Using Caenorhabditis elegans,
we demonstrate that multiple plasma membrane endocytic adaptors
function redundantly to regulate clathrin-mediated endocytosis and
to recruit components of the endosomal sorting complex required for
transport (ESCRT) machinery to the cell surface to direct the sorting of
ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the
endolysosomal system. In the absence of a heterooligomeric adaptor
complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of
its clathrin-mediated internalization. Consistent with a role for the
ESCRT machinery during cargo endocytosis, we further show that the
ESCRT-0 complex accumulates at a subset of clathrin-coated pits on
the surface of human cells. Our findings suggest a unique mechanism
by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.
Fig. 1. FCHO-1, EHS-1, and ITSN-1 are plasma membrane-specific adaptor
proteins. (A) Whole-worm extracts generated from control (N2) animals and
mutant strains were separated by SDS/PAGE and immunoblotted by using
affinity-purified antibodies as indicated. (B) Embryos isolated from control
hermaphrodites (N2) and strains lacking FCHO-1 (Top Right) or ITSN-1
(Bottom Right) were fixed and stained by using antibodies directed against
each protein. (C) Control embryos were fixed and stained by using antibodies directed against RAB-5 (Cy2; Left) and ITSN-1 (Cy3; Center). A 7×
zoom of a boxed region is also provided (Right). (D) Control (Upper) and
VPS-32–depleted embryos (Lower) stably expressing a GFP fusion to clathrin
light chain (GFP-CLIC-1) were imaged by using swept-field confocal optics.
Maximum intensity projections of four cortical sections (2 μm thick), taken
∼9 μm from the coverslip, are shown. (E) Following background subtraction,
the total integrated intensity of GFP-CLIC-1 at the cell-cell junction of twocell stage embryos was calculated (within several 1-μm2 regions) in control
and vps-32(RNAi) embryos. Representative images are shown (n = 12 embryos for each condition). **, significant difference (P < 0.01). (F) Embryos
expressing GFP-CAV-1 were depleted of VPS-32 by using RNAi and subsequently fixed and stained by using antibodies directed against ITSN-1 and
GFP. (Scale bars: 10 μm.)
the restricted localization of these endocytic adaptors during early
embryonic development in worms, we analyzed their distribution
in embryos depleted of an ESCRT component (VPS-32), which
causes a dramatic alteration in endosome morphology and leads
to the aberrant hyperaccumulation of proteins that only transiently associate with endosomal membranes under normal conditions (Fig. S2 C and D; refs. 21 and 22). Although inhibition of
ESCRT function resulted in a substantial redistribution of clathrin light chain from the plasma membrane (Fig. 1 D and E),
localization of the endocytic adaptors was unaffected (Fig. 1F and
Fig. S2E). Based on these collective findings, we conclude that
FCHO-1, EHS-1, and ITSN-1 are enriched on the plasma membrane but largely absent from endosomes.
The FCHO proteins have been suggested to act as major interaction hubs, which bind to multiple factors that contribute to
clathrin-mediated endocytosis, including Eps15 and intersectin (7–
9, 11, 13). To determine whether native FCHO-1 associates stably
with EHS-1 and ITSN-1, we separated a C. elegans embryo extract
by using size exclusion chromatography and determined the elution profiles of each endocytic adaptor. We found that FCHO-1
cofractionated with both EHS-1 and ITSN-1 in two unique peaks,
suggesting that the three proteins coassemble into at least two
distinct complexes in vivo (Fig. 2 A and B). Interestingly, the larger
11858 | www.pnas.org/cgi/doi/10.1073/pnas.1302918110
Fig. 2. FCHO-1, EHS-1, and ITSN-1 form a stable complex that functions in
the trafficking of ubiquitin-modified cargoes to the lysosome for degradation. (A) An embryo extract was separated by gel filtration chromatography,
and eluted fractions were immunoblotted by using antibodies directed
against ITSN-1, FCHO-1, EHS-1, and APA-2. Based on densitometry measurements, peak fractions were identified for each protein (boxed regions).
(B) Graphical representation of densitometry measurements conducted on
immunoblots shown in A. (C) Recombinant ITSN-1, EHS-1, and FCHO-1 were
applied individually or as a mixture onto a gel filtration column, and eluted
fractions were separated by SDS/PAGE before silver staining. The Stokes
radius for each protein was calculated based on the elution profiles of
characterized standards. (D) Recombinant ITSN-1, EHS-1, and FCHO-1 were
applied individually or as a mixture onto glycerol gradients (10–30%), which
were centrifuged and fractionated. Each fraction was separated by SDS/
PAGE and silver stained. Densitometry measurements, which are shown in
a graphical representation, were used to define the sedimentation value of
each protein (boxed region). (E) Immobilized control, ehs-1;itsn-1;fcho-1
triple mutant animals, and stam-1 single mutant animals expressing GFPCAV-1 were imaged by using DIC (Left) and swept-field confocal optics
(Right). Cartoons highlighting the number of cells contained within each
embryo observed in utero are provided (Center). Additionally, a 1.6× zoom
of a boxed region within the uterus of each animal is also shown. (F) The
rates of GFP-CAV-1 endocytosis from the plasma membrane following ovulation were analyzed in control (N2) or ehs-1;itsn-1;fcho-1 triple mutant
embryos by measuring the loss of cell surface fluorescence over time in utero
(n = 5 for each condition). In some cases, animals were depleted of epsin
(epn-1), APA-2, or clathrin heavy chain (chc-1) as indicated. A comparison of
GFP:CAV-1 localization in one cell stage ehs-1;itsn-1;fcho-1 triple mutant
embryos (during metaphase) following APA-2 or EPN-1 depletion is also
shown (Right). (Scale bars: 10 μm.)
peak also contained subunits of the AP-2 complex, which has been
shown to associate with Eps15, intersectin, and FCHO1 (7, 8).
Thus, our data indicate that FCHO-1, EHS-1, and ITSN-1 form
a complex that is capable of existing independently or in association with the AP-2 complex.
Mayers et al.
The FEI Adaptor Complex Regulates Protein Sorting in the Endolysosomal
Pathway. Endocytic adaptors play a key role in cargo selection (5, 6,
15, 16, 24). To investigate a potential role for components of the
FEI complex in ubiquitin-dependent protein sorting in C. elegans,
we used previously isolated strains containing deletion mutations in
each gene (Fig. S1B). In contrast to the essential roles of the FCHO
proteins, Eps15, and intersectin in other species (7, 8, 25), null
mutations in each gene failed to substantially impact C. elegans viability or embryonic development (Table S1).
We next analyzed the effect of each loss-of-function mutation
on the trafficking of CAV-1, the C. elegans homolog of caveolin-1,
which was shown to undergo clathrin-mediated endocytosis and
ubiquitin-dependent transport to the lysosome for degradation
(26, 27). In the proximal oocytes of control animals, CAV-1
resides on Golgi-derived cortical granules, which fuse en masse
with the plasma membrane following fertilization and ovulation.
CAV-1 is subsequently internalized and ubiquitin-modified during the one-cell stage of embryonic development and rapidly
degraded in an ESCRT-dependent manner (Fig. 2E and Fig. S2F;
refs. 21 and 26). Individual loss of function mutations in FCHO-1,
EHS-1, or ITSN-1 failed to elicit a defect in CAV-1 trafficking
and degradation (Fig. S2G). To determine whether components
of the FEI complex may function redundantly in cargo sorting, we
generated mutant strains lacking multiple components. We found
that animals lacking all three subunits exhibited a significant delay
in CAV-1 degradation and enhanced embryonic lethality compared with the single and double mutant strains (Fig. 2E, Fig.
S2G, and Table S1). Importantly, this effect was not a result of
disrupted clathrin or AP-2 recruitment to the plasma membrane
(Fig. S2 H and I). Instead, these data suggest that FCHO-1, EHS1, and ITSN-1 directly regulate the sorting of ubiquitin-modified
cargoes and can partially substitute for one another during early
C. elegans development. Additionally, our findings argue against
a model in which FCHO proteins act as nucleating factors for
clathrin-mediated endocytosis.
A delay in CAV-1 degradation could arise from either impaired endocytosis at the plasma membrane or a subsequent
defect in cargo sorting to the lysosome. To discriminate between
these possibilities, we analyzed the rate of GFP-CAV-1 internalization based on changes in its fluorescence intensity at the
plasma membrane following ovulation. Surprisingly, we found
that embryos lacking all three FEI subunits did not exhibit
a significant defect in CAV-1 endocytosis compared with control
eggs (Fig. 2F and Movies S1 and S2). Instead, CAV-1 remained
associated with Rab5-positive early endosomes for an extended
period in mutant embryos, which was not observed in control
animals (Fig. S3A), but was reminiscent of the defect observed in
animals lacking the function of one or more components of the
ESCRT machinery (Fig. 2E; ref. 21).
To determine whether alternative endocytic factors function in
CAV-1 endocytosis, we individually depleted all known C. elegans
clathrin adaptors, including homologs of human stonin (UNC41), AP180/CALM (UNC-11), the alpha subunit of AP-2 (APA2), Disabled (DAB-2), NUMB (NUM-1), and epsin (EPN-1) and
examined trafficking of CAV-1 (Fig. S3B). Although we failed to
identify changes in the rate of CAV-1 internalization following
Mayers et al.
individual depletion of these components in otherwise wild-type
animals, we found that inhibition of EPN-1 in animals lacking all
components of the FEI complex specifically caused a potent inhibition of CAV-1 endocytosis, similar to that observed following
depletion of clathrin heavy chain (Fig. 2F and Fig. S3B). Collectively, these data indicate that subunits of the FEI complex act
redundantly to regulate multiple steps of cargo sorting at the
plasma membrane and potentially recruit additional adaptors that
function in the endolysosomal trafficking of ubiquitin-modified
membrane proteins.
In addition to our analysis of CAV-1, we further defined the role
of the FEI complex in the trafficking of MIG-14, a characterized
clathrin-dependent cargo that recycles instead of being degraded
in the lysosome and, thus, is unlikely to be ubiquitin-modified (28).
During fertilization and ovulation, MIG-14 is largely internalized
in control embryos (Fig. S3 C and D and Movie S3). However, in
embryos lacking all components of the FEI complex, significantly
higher levels of MIG-14 are observed on the plasma membrane
(Fig. S3 C and D and Movie S4), although its endocytosis is not
entirely blocked. These data further support our findings demonstrating that the FEI complex acts redundantly with other adaptors
during cargo internalization, likely together with AP-2 in this case,
which was implicated in MIG-14 endocytosis (29). Importantly,
movement of MIG-14 through endosomal compartments was not
delayed in the triple fcho-1;ehs-1;itsn-1 mutant strain, consistent
with the idea that the function of the FEI complex is limited to the
cell surface (Fig. S3 D and E).
The FEI Complex Interacts Directly with ESCRT-0 at the Plasma Membrane.
To identify additional cargo adaptors that function together with
the FEI complex, we took advantage of antibodies directed against
EHS-1 and ITSN-1 to conduct a series of immunoprecipitations
from C. elegans embryo extracts. Using solution mass spectrometry
and immunoblot analysis, we reproducibly isolated a set of factors
known to function during endocytic protein transport, including all
components of the FEI complex, clathrin heavy and light chains,
members of the AP-2 adaptor complex, and both subunits of the
ESCRT-0 complex (STAM-1 and HGRS-1; Fig. S4A and Table
S2). Reciprocal immunoprecipitations using STAM-1 and HGRS-1
antibodies confirmed the existence of this interaction network (Fig.
S4A and Table S2). Additionally, we found that a subpopulation of
native STAM-1 and HGRS-1 cofractionate with the AP-2 and FEI
complexes following size exclusion chromatography of a C. elegans
embryo extract (Fig. S4B). Notably, we failed to observe associations between components of the FEI complex and other ESCRT
subunits, suggesting that the interaction with ESCRT-0 was specific
(Table S2). These data indicate that the FEI complex, potentially in
concert with AP-2 and clathrin, binds to ESCRT-0.
Previous studies suggest that mammalian ESCRT-0 binds
specifically to a short isoform of Eps15 (Eps15b) that lacks
amino-terminal EH domains, localizes only to endosomes, and
does not interact strongly with the AP-2 complex (30). In contrast, our data indicate that C. elegans ESCRT-0 interacts with
a plasma membrane isoform of EHS-1 that also coimmunoprecipitates with components of the AP-2 complex. To further validate the interaction, we generated GST fusions to three partially
overlapping regions of EHS-1 and tested their ability to associate
with native ESCRT-0. Our findings demonstrated that the aminoterminal EH domains of EHS-1 bound specifically to ESCRT-0 (Fig.
S4C). We confirmed this association using the GST fusions and
recombinant STAM-1, indicating that EHS-1 binds directly to the
ESCRT-0 complex (Fig. S4D). To determine whether ITSN-1 and
FCHO-1 also associate with ESCRT-0, we generated recombinant
fragments of each protein for interaction studies. We found that
both the coiled-coil and SH3 domains of ITSN-1 bound to
ESCRT-0 directly (Fig. S4 E and F). Additionally, we found that
the middle and μ-homology domains of FCHO-1 bound to
ESCRT-0 under similar conditions (Fig. S4F). Together, these
studies indicate that multiple components of the FEI complex
associate with ESCRT-0.
PNAS | July 16, 2013 | vol. 110 | no. 29 | 11859
CELL BIOLOGY
To confirm that FCHO-1, EHS-1, and ITSN-1 assemble into
a stable complex, we expressed each full-length protein recombinantly and assayed their ability to bind to one another. Individually, each adaptor protein exhibited a distinct elution
profile following gel filtration chromatography (Fig. 2C). However, when mixed together, the three proteins coeluted as a single complex with an average Stokes radius of 9.5 nm (Fig. 2C).
They also remained associated during sedimentation velocity
centrifugation, exhibiting an S value of 10.8 S (Fig. 2D). Based
on its hydrodynamic properties (23), we estimated the native
molecular mass of the complex to be ∼430 kDa. Together, these
data indicate that FCHO-1, EHS-1, and ITSN-1 form a stable
heterooligomeric complex on the plasma membrane, which we
refer to as the FEI adaptor complex.
Our data indicate that the FEI complex localizes to the plasma
membrane, suggesting that an association with ESCRT-0 would
be restricted to that site. To investigate this possibility, we conducted immunofluorescence studies using HGRS-1 and STAM-1
antibodies. Consistent with previous findings (31), imaging of the
one-cell stage embryo (away from the cell surface) revealed that
both components of ESCRT-0 localize to endosomes (Fig. 3A).
However, we additionally observed an accumulation of each
protein at or near the plasma membrane, which was most obvious in multicellular embryos (Fig. 3B). In contrast, we failed to
observe a similar cell surface localization of other endosomal
proteins, including EEA-1 (Fig. 3C). To determine whether the
cell surface distribution of ESCRT-0 depends on the presence of
the plasma membrane-associated endocytic adaptor proteins, we
examined its localization in a variety of mutant backgrounds.
Strikingly, in triple mutant animals lacking all components of the
FEI complex, ESCRT-0 localization to the plasma membrane
was dramatically reduced, although not completely abolished
(Fig. 3D and Fig. S5A). Because the ESCRT-0 plasma membrane interaction network also included the AP-2 complex, we
examined the effect of depleting APA-2 by using RNA interference (RNAi) in control animals. Under these conditions,
we observed a relatively minor diminution in ESCRT-0 localization at the cell surface (Fig. 3D). To determine whether the AP-2
and FEI complexes function redundantly in targeting ESCRT-0,
we depleted APA-2 in animals lacking FCHO-1, EHS-1, and
ITSN-1. In the absence of these four components, we observed
a dramatic defect in ESCRT-0 recruitment to the plasma membrane (Fig. 3D). Additionally, embryonic viability was reduced significantly under these conditions, highlighting the overlapping roles
of the FEI and AP-2 complexes during C. elegans embryogenesis (Table S1). Taken together, these data strongly suggest that multiple endocytic adaptor complexes function to recruit
ESCRT-0 to the cell surface to engage ubiquitin-modified substrates
at the plasma membrane.
A major determinant for ESCRT-0 localization is its affinity for
phosphatidylinositol 3-phosphate (PI3P), which is enriched on early
endosomes (32). However, additional anionic phospholipids also
participate in targeting FYVE domain-containing proteins (33). To
determine whether the ESCRT-0 complex can also bind to membranes that are devoid of PI3P, we conducted a series of liposome
coflotation assays. We found that the presence of 15% phosphatidylserine (PS) in liposomes, similar to the concentration of negatively
charged phospholipids in the plasma membrane (34), is sufficient to
promote membrane association of ESCRT-0 (Fig. 4A). These data
suggest that a combination of lipid- and protein-dependent associations can promote ESCRT-0 localization to the cell surface.
ESCRT-0 Associates with Clathrin-Coated Pits in Human Cells. Based
on its interactions with endocytic adaptor proteins at the plasma
membrane, we investigated the possibility that ESCRT-0 is
recruited specifically to sites of clathrin-mediated endocytosis.
To do so, we used total internal reflection fluorescence (TIRF)
microscopy to visualize both clathrin and ESCRT-0 at the cell
surface of mammalian tissue culture cells. Following transient
transfection of a construct encoding RFP-clathrin light chain
(RFP-LCa) into HeLa cells stably expressing a low level of YFPHrs, we identified numerous sites of clathrin-coated pit formation on the plasma membrane (Fig. 4B). Consistent with previous
work (10), some of these sites exhibited a short lifetime (in the
60- to 90-s range), whereas others persisted for several minutes.
Two-color TIRF imaging revealed that YFP-Hrs colocalized
with a subpopulation (∼27%) of clathrin-coated structures (Fig.
4B; n = 641 sites analyzed). Importantly, overexpression of YFPHrs that was sufficient to alter endosome morphology (Fig. S5B)
did not significantly increase the percentage of clathrin-coated
pits that exhibited the presence of ESCRT-0. Time-lapse analysis
further demonstrated that Hrs accumulation at the plasma
membrane did not precede or coincide with the initial arrival of
clathrin light chain (Fig. 4 C and D). However, disappearance of
Hrs occurred simultaneously with the movement of clathrin out
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Fig. 3. ESCRT-0 localization to the plasma membrane depends on the
presence of multiple endocytic adaptors. (A) One cell stage embryos were
fixed and stained with directly labeled antibodies against HGRS-1 (Cy2) and
EEA-1 (Cy3). Images (∼4 μm from the coverslip) were acquired by using
swept-field confocal optics. A 3× zoom of the boxed region is also shown
(Right). (B and C) Four cell-stage embryos were fixed and stained with labeled antibodies against HGRS-1 (Cy2), EEA-1 (Cy2), or ITSN-1 (Cy3). Images
(∼9 μm from the coverslip) were acquired by using swept-field confocal
optics. A 3× zoom of boxed regions is also provided (Right). (D) Four cell
stage embryos expressing a GFP fusion to a CAAX motif (Fig. S5A) were fixed
and stained by using antibodies directed against HGRS-1, and plasma
membrane localization was quantified based on fluorescence intensity by
using a linescan analysis (highlighted in each image). Peak fluorescence intensity of the GFP signal was used to define the localization of the plasma
membrane. A table showing the relative intensity of HGRS-1 on the plasma
membrane in different mutant backgrounds is shown on Right (++, high
level of HGRS-1; +, low level of HGRS-1; −, an absence of HGRS-1). (Scale
bars: A–D, 10 μm; A–C Inset, 2 μm.)
of the evanescent field, strongly suggesting that ESCRT-0 continues to associate with clathrin-coated vesicles throughout
the scission process (Fig. 4 C and D). In contrast to the accumulation of ESCRT-0 at some clathrin-coated pits, we failed to
observe the downstream ESCRT-I complex at these sites (Fig.
4E), consistent with the specific biochemical associations we
identified among the ESCRT-0, FEI, and AP-2 complexes at
the plasma membrane.
To further investigate whether ESCRT-0 accumulates at cargoladen coated pits, we pulse labeled cells that were serum starved
overnight with fluorescently labeled epidermal growth factor
(EGF), followed by fixation. Cells were also pretreated with the
dynamin inhibitor dynasore for 1 h before the brief (2 min) EGF
stimulation, to ensure that membrane-associated EGF was restricted to the cell surface (Fig. S5C). Under these conditions, we
found that YFP-Hrs accumulated at a subpopulation (∼14%) of
EGF-labeled sites (Fig. 4F; n = 564 sites analyzed). Collectively,
our findings highlight a role for ESCRT-0 as an additional plasma
membrane endocytic adaptor complex that functions during
a subset of clathrin-mediated endocytosis events (Fig. 4G).
Mayers et al.
Discussion
Ubiquitin modification of transmembrane cargoes plays a key
role in directing their entry into the endolysosomal system. At
clathrin-coated pits, both Eps15 and epsin, which harbor motifs
that associate weakly with ubiquitin, have been suggested to bind
cargoes and potentially concentrate them before endocytosis (5).
How these cargoes are then transferred efficiently to other ubiqutin-binding proteins at endosomes is difficult to envision. Our
findings suggest that an alternative pathway exists to alleviate the
need to resort cargoes following their arrival at endosomes.
Specifically, we found that a component of the ESCRT machinery
can associate directly with clathrin-coated pits, allowing it to
engage ubiquitin-modified cargoes before their entry into the
endosomal system. Furthermore, our data show that ESCRT-0 is
present throughout the scission process, suggesting that cargoes
remain bound to ESCRT-0 following internalization. In contrast,
Eps15 and epsin arrive early to recruit and/or sequester ubiquitinmodified cargoes at clathrin-coated pits (10). Before internalization,
ESCRT-mediated capture facilitates continued cargo clustering
during vesicle transport and subsequent endosome fusion. Thus,
preassembly of ESCRT-0 at nascent pits would enhance the
Mayers et al.
Materials and Methods
Antibodies, Immunofluorescence, and Live Imaging. C. elegans FCHO-1, EHS-1,
and ITSN-1 antibodies were raised in rabbits, which were immunized with
either a GST fusion to a fragment of FCHO-1 (amino acids 1–274), a GST
fusion to a fragment of ITSN-1 (amino acids 1–283), or a polyhistidine-tagged form of full length EHS-1 produced in Escherichia coli and affinity purified. Antibodies directed against HGRS-1, STAM-1, EEA-1, APA-2, and RAB-5
have been described (40, 41). Confocal images were acquired on a sweptfield confocal microscope (Nikon Ti-E), and TIRF imaging was conducted on
a Nikon TE2000E equipped with a TIRF illuminator. Acquisition parameters
and image analysis (including linescan analysis and the creation of kymographs) was performed by using Nikon Elements (confocal) or Metamorph
(TIRF) software. Immunofluorescence of fixed embryos was performed as
described (42) by using directly labeled rabbit antibodies at a concentration
of 1 μg/mL 30–40 Z sections at 0.2-mm steps were acquired (depending on
sample thickness). To calculate the fluorescence intensity of specific proteins,
PNAS | July 16, 2013 | vol. 110 | no. 29 | 11861
CELL BIOLOGY
Fig. 4. ESCRT-0 associates with clathrin-coated pits at the plasma membrane.
(A) Coflotation assays were conducted by using recombinant ESCRT-0 and
liposomes containing either 70% phosphatidylcholine (PC) and 30% phosphatidylethanolamine (PE); 69% PC, 30% PE, and 1% PI3P; or 15% PS, 55%
PC, and 30% PE. Asterisks highlight the presence of bacterial contaminants.
(B) HeLa cells expressing RFP-clathrin light chain (LCa) and YFP-Hrs were imaged by using TIRF microscopy. (C) Individual sites of clathrin-mediated endocytosis were imaged over time by using TIRF microscopy. Merged panels
showing both RFP-LCa and YFP-Hrs localization are shown (Bottom). (D)
Kymographs showing the fate of RFP-LCa and YFP-Hrs at clathrin-coated pits
over time. Arrows highlight the timing of Hrs appearance (Left) or disappearance (Right). (E) HeLa cells expressing RFP-clathrin light chain (LCa) and
GFP-Mvb12B were imaged by using TIRF microscopy. (F) HeLa cells stably
expressing YFP-Hrs were pretreated with dynasore (200 μM) for 1 h and
subsequently pulse-labeled with fluorescent EGF (10 ng/mL) for 2 min before
fixation and imaging. (G) Model highlighting a role for ESCRT-0 in the capture of ubiquitin-modified cargoes at a subset of clathrin-coated pits. (Scale
bars: B and E, 10 μm; C and D, 2 μm; F, 5 μm.)
efficiency by which cargoes are sorted and, thereby, accelerate
downstream trafficking steps, including ubiquitin-dependent degradation in lysosomes.
The role for ESCRT-0 at the cell surface may be similar to
that of other factors that associate with clathrin-coated pits, but
function at later steps of membrane transport. For example, the
RME-6 exchange factor for Rab5 was shown previously to bind
to the APA-2 subunit of the AP-2 complex. Although unlikely to
function directly in vesicle formation, the presence of RME-6 at
clathrin-coated pits likely enhances Rab5 activity, which is necessary for vesicle fusion with the endosome (35). In an analogous
fashion, RME-4 also binds to APA-2 and activates Rab35, which
functions later in cargo recycling (36). In Drosophila oocytes,
components of the exocyst complex also associate with clathrincoated pits (37). Although the association is dispensable during
receptor internalization, the exocyst plays a key role in receptor
recycling following endocytosis. Likewise, ESCRT-0 is unlikely
to influence the process of clathrin-mediated endocytosis directly. Indeed, our data demonstrated that perturbations, which
inhibit ESCRT-0 recruitment to the plasma membrane, fail to
affect the rate of internalization of ubiquitin-modified cargoes.
However, subsequent steps of cargo sorting were dramatically
delayed. Collectively, these findings suggest that in addition to
their specific roles in clathrin-mediated endocytosis, endocytic
adaptor proteins also promote key priming events that prepare
cargoes for downstream sorting.
Unlike the majority of clathrin adaptors studied to date,
ESCRT-0 appears to associate with only a subpopulation of coated
pits in human cells. Although its presence at additional sites of
endocytosis may be below our detection limit, these data highlight
the idea that all clathrin-coated pits are not uniform in composition. Instead, they may be strongly influenced by the presence of
additional factors, such as cargo molecules (38). In particular,
recent work suggests that ubiquitin can directly influence the
maturation of a coated pit by acting as a signaling molecule in
a dose-dependent fashion. Interestingly, the plasma membrane
distribution of the mu opioid receptors, which undergo ubiquitin
modification upon agonist stimulation, does not overlap uniformly
with clathrin when activated, supporting the idea that multiple,
distinct populations of clathrin-coated pits exist (39). These findings also raise the possibility that ubiquitin-mediated signaling
during pit maturation is coupled to ESCRT-0 recruitment. Because membrane-associated ESCRT-0 is capable of binding at
least eight ubiquitin molecules simultaneously (40), the localized
enrichment of ubiquitin-modified cargoes at a subset of coated
pits may help to specify the timing and/or site of its recruitment.
Furthermore, the additional presence of endocytic adaptors,
acidic phospholipids, and clathrin, all of which can associate
with ESCRT-0, may further promote a level of coincidence
detection that is necessary to enhance targeting to sites of
clathrin-mediated endocytosis. This type of integration is
likely important for the rapid and specific clearance of ubiquitin-modified cargoes, which is observed following receptor
stimulation in human cells, as well as major changes to plasma
membrane content following fertilization in C. elegans.
the total intensity in a box containing the signal (from a maximum intensity
projection) was measured and the camera background was subtracted. For
live imaging of C. elegans, animals were immbolized and mounted on an
agarose pad. Alternatively, embryos were dissected from gravid animals and
mounted for imaging. HeLa cells were grown on 35-mm glass bottom dishes
maintained at 37 °C for time-lapse TIRF imaging. Treatments with dynasore
were conducted as described (43).
Worm Strains, RNA Interference, and Cell Culture. All C. elegans strains were
derived from the Bristol strain N2. The generation of animals expressing
fluorescent fusions with CAV-1, MVB-12, and clathrin light chain were described (26, 42). Double-stranded RNA (dsRNA) was synthesized as described
(42) from templates prepared by using specific primers to amplify N2 genomic
DNA. For RNAi experiments, early L4 stage hermaphrodites were soaked in
dsRNA for 24 h at 20 °C within a humidified chamber. Animals were allowed
to recover for 48 h before analysis. HeLa cells were maintained in DMEM
supplemented with 10% (vol/vol) FBS (HeLa), penicillin/streptomycin, and
L-glutamine at 37 °C in the presence of 5% CO2. Cells stably expressing a low
level of YFP-Hrs were generated by retroviral infection, followed by fluorescence-activated cell sorting to isolate clonal populations, which were further
sorted manually. Transfections were conducted by using FuGENE HD.
lysates were generated for immunoprecipitations as described (42). For mass
spectrometry, proteins were precipitated by using TCA and were subsequently processed for MudPIT analysis (42). The spectra were searched
with either the SEQUEST or ProLuCID (44) algorithm against the WormBase
C. elegans database.
Recombinant protein expression was performed by using BL21 (DE3)
E. coli, and purifications were conducted by using either glutathione
agarose beads (for GST fusions) or nickel affinity resin (for polyhistidinetagged proteins). Hydrodynamic studies and immunoblotting of extracts
and immunoprecipitates were performed as described (42). To determine
whether endogenous ESCRT-0 could interact with various fragments of
recombinant EHS-1, ITSN-1, or FCHO-1, extracts were generated from embryos that were subsequently subjected to sonication in lysis buffer.
Extracts were clarified by centrifugation (100,000 × g) before incubation
with proteins bound to glutathione affinity resin. Coflotation assays were
conducted as described (41).
Mass Spectrometry, Biochemistry, and Liposome Flotation Assays. Adult hermaphrodites were grown synchronously in liquid culture, and embryonic
ACKNOWLEDGMENTS. We thank Sun Lee for technical assistance. We also
thank the Caenorhabditis Genetics Center and the National Bioresource
Project (Japan) for sharing C. elegans mutant strains and members of the
A.A. laboratory for critically reading this manuscript. This work was supported by National Institutes of Health Grants 1R01GM088151-01A1 (to A.A.)
and P41RR011823 (to J.R.Y.).
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Supporting Information
Mayers et al. 10.1073/pnas.1302918110
Fig. S1. The domain organization of human and Caenorhabditis elegans Eps15, intersectin, and FCHO proteins are conserved. (A) Cartoons depicting the
domain architecture of the C. elegans and human isoforms of Eps15, intersectin, and FCHO proteins. CC, coiled-coil domain; DPF, Aspartic acid-proline-phenylalanine rich domain; EFC/F-BAR, Extended FCH homology domain/FCH-BAR domain; EH, Eps15-homology domain; μ-HD, mu homology domain; SH3, Src
homology 3 domain. (B) Predicted gene models for ehs-1, itsn-1, and fcho-1. Exons and introns are shown to scale. Deletion mutations (highlighted in red) and
a brief description of the predicted effect of each are provided.
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Fig. S2. The components of the FEI complex (FCHO-1, EHS-1, and ITSN-1) localize specifically to the plasma membrane and do not accumulate on endosomes.
(A) Four cell stage control and ehs-1 mutant embryos were fixed and stained by using antibodies directed against EHS-1 and visualized by using swept-field
confocal optics. An arrow highlights the plasma membrane localization of EHS-1. Nonspecific staining in the P1 cell is present in both control and ehs-1 mutant
embryos. (B–D) Two-cell stage control (B and C) or VPS-32 depleted (D) embryos expressing GFP-DYN-1 (B) or GFP-MVB-12 (C and D) were fixed and stained by
using antibodies directed against ITSN-1 and GFP (B and D) or imaged live (C). Embryos were imaged by using swept-field confocal optics. (E) Two cell stage,
VPS-32 depleted embryos were fixed and stained by using antibodies directed against ITSN-1 and HGRS-1. Embryos were imaged by using swept-field confocal
optics. (F) Cartoon depicting the domain architecture of a C. elegans isoform of caveolin-1 (CAV-1). Two lysine residues in the amino-terminal cytosolic region
of CAV-1 were identified as potential sites for ubiquitin modification (K49 and K74), based on mass spectrometry analysis of a CAV-1 immunoprecipitate
(isolated specifically from an embryo extract). For this experiment, we immunoprecipitated GFP:CAV-1 (in the presence of N-ethylmaleimide) by using GFP
antibodies that were coupled to Protein A agarose. Following a low pH elution, CAV-1 was TCA precipitated, washed extensively with acetone, and dried by
using vacuum centrifugation. The pellet was solubilized, and disulfide bonds were reduced. Free thiols were then alkylated, and the sample was digested
sequentially with Endoproteinase Lys-C and trypsin. The reaction was quenched after 12 h, and samples were pressure loaded onto a fused silica capillary
desalting column, which was placed inline with a Hewlett Packard Agilent 1100 Quaternary Pump and analyzed by using a customized four-step separation
method (90, 120, 120, and 150 min, respectively). A distal 2.5-kV spray voltage was applied to elute the peptides from the microcapillary column. This applied
voltage caused the peptides to directly electrospray into a linear trap quadrupole (LTQ) two-dimensional ion trap mass spectrometer. For each step of the
multidimensional cycle, one full-scan mass spectrum (400–2,000 m/z) occurred followed by five data-dependent MS/MS spectra at a 35% normalized collision
energy. Spectra were searched against a recent version of the C. elegans protein database by using the SEQUEST algorithm. The search was set up to consider
a possible modification of +114 on lysines, which corresponds to the two glycine residues from ubiquitin that remain attached to a target lysine after trypsinization. The program DTASelect was used to filter peptide identifications and manually validate spectra that correspond to putative ubiquitin attachment
sites. (G) Table illustrating the defects observed in GFP:CAV-1 degradation a variety of strains, as indicated. Embryos containing at least 15% of the GFP
fluorescence intensity observed immediately following ovulation were counted as being fluorescent (n = 15 animals each). (H) Control and fcho-1; ehs-1; itsn-1
triple mutant animals expressing a GFP fusion to clathrin light chain (CLIC-1) were imaged by using swept-field confocal optics. Loss of all components of the FEI
complex fails to alter the enrichment of clathrin on the plasma membrane of oocytes in the C. elegans germ line. (I) Control, fcho-1; ehs-1; itsn-1 triple mutant,
and apa-2 single mutant embryos were fixed and stained by using an antibody directed against endogenous APA-2 (the alpha subunit of the AP-2 complex)
and imaged by using swept-field confocal optics. Loss of all components of the FEI complex fails to alter the enrichment of AP-2 on the plasma membrane of
embryos. Intracellular fluorescence observed in apa-2 mutant embryos reflects nonspecific staining. (Scale bars: 10 μm.)
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Fig. S3. Inhibition of FEI complex function affects endocytosis in a cargo-selective fashion. (A) Four cell stage control and ehs-1;itsn-1;fcho-1 triple mutant
animals expressing GFP:CAV-1 were fixed and staining by using antibodies directed against GFP and RAB-5. Embryos were imaged by using swept-field confocal
optics. Inset showing a 2× zoom of the boxed regions is provided. Arrows highlight endosomes that exhibit both CAV-1 and RAB-5 staining. (B) Table indicating
the impact of various double-stranded RNAs (dsRNAs) on the viability (through early larval development) and internalization rate of GFP-tagged CAV-1 in control
and fcho-1; ehs-1; itsn-1 triple mutant embryos. (C) Bar graph showing the relative fluorescence intensities (following background subtraction) of cell surface
MIG-14:GFP during metaphase of the first embryonic division in control and fcho-1;itsn-1 double mutant animals (treated with dsRNA targeting EHS-1). **,
statistically significant difference compared with control (P < 0.01). (D) Control and fcho-1;itsn-1 double mutant animals expressing MIG-14:GFP were analyzed in
utero by using DIC and swept-field confocal optics. The mutant animals were treated with dsRNA targeting EHS-1 to better mimic the genetic perturbation of
ehs-1;itsn-1;fcho-1 triple mutant animals. The embryos shown were tracked until metaphase of the first embryonic division to highlight differences in MIG-14:GFP
localization under each condition. We failed to observe any increase in MIG-14:GFP accumulation on endosomes following inhibition of the FEI complex, regardless of embryonic stage. (E) Bar graph showing the relative fluorescence intensities (following background subtraction) of endosomal compartments
containing MIG-14:GFP during metaphase of the first embryonic division in control and fcho-1;itsn-1 double mutant animals (with or without additional
treatment with dsRNA targeting EHS-1). **, statistically significant difference compared with control (P < 0.01). (Scale bars: A and D, 10 μm; Inset, 2.5 μm.)
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Fig. S4. Components of the FEI complex bind to ESCRT-0. (A) Antibodies directed against GFP (Mock), EHS-1, STAM, and HGRS-1 were used to immunoprecipitate each protein and interacting partners from C. elegans embryo extracts. Immunoprecipitates were immunoblotted for the presence of HGRS-1, STAM,
EHS-1, and ITSN-1. (B) An embryo extract was separated by gel filtration chromatography, and eluted fractions were immunoblotted by using antibodies directed
against HGRS-1, STAM-1, FCHO-1, and APA-2. Based on densitometry measurements, peak fractions were identified for each protein (boxed regions). (C) Cartoon
diagrams highlighting the various regions of EHS-1 that were purified as GST fusion proteins for interaction studies. (D–H) GST and various domains of EHS-1,
ITSN-1, and FCHO-1 (see cartoons) fused to GST were immobilized on glutathione agarose beads and incubated with C. elegans embryo extracts or recombinant
STAM-1 (rSTAM-1). The eluted fractions from the beads were immunoblotted for the presence of HGRS-1, STAM-1 (or rSTAM-1), EHS-1, and ITSN-1.
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Fig. S5. ESCRT-0 is recruited to the plasma membrane. (A) Embryos expressing a GFP fusion to a CAAX box (GFP:CAAX) were stained by using antibodies
directed against endogenous HGRS-1 and GFP and imaged by using swept-field confocal optics. A 1.6× zoom of the boxed region indicated is shown as a color
overlay (Right). (B) HeLa cells overexpressing YFP-tagged Hrs were imaged by using swept-field confocal optics. (C) HeLa cells pretreated with dynasore were
pulse labeled with EGF (directly labeled with Alexa-647) for 2 min, followed by fixation and imaging by using swept-field confocal optics. A medial section of
a representative cell is shown. Under these conditions, EGF is restricted to the cell surface. (Scale bars: 10 μm.)
Table S1. Viability of embryos lacking one or more endocytic
adaptor proteins
fcho-1
N2
APA-2 depletion
Embryos analyzed, n
Viability, %
ehs-1;
itsn-1
ehs-1;
itsn-1;
fcho-1
−
+
−
+
−
+
−
+
529
100
1,096
100
645
93
791
92
982
100
1,507
97
400
55
779
28
Table S2. Identification of EHS-1, ITSN-1, STAM-1, and HGRS-1 interacting partners using immunoprecipitation
followed by solution mass spectrometry
Sequence coverage, %
C. elegans gene
stam-1 (C34G6.7)
hgrs-1 (C07G1.5)
chc-1 (T20G5.1)
clic-1 (T05B11.3)
itsn-1 (Y116A8C.36)
ehs-1 (ZK1248.3)
fcho-1 (F56D12.6)
apa-2 (T20B5.1)
apb-1 (Y71H2B.10)
dpy-23 (R160.1)
tsg-101 (C09G12.9)
Human homolog(s)
EHS-1 (IP)
ITSN-1 (IP)
STAM-1 (IP)
HGRS-1 (IP)
Predicted size, kDa
STAM (ESCRT-0)
Hrs (ESCRT-0)
Clathrin Heavy Chain
Clathrin Light Chain
Intersectin
Eps15
FCHO1/2
AP-2α subunit
AP-2β subunit
AP-2μ subunit
Tsg101 (ESCRT-I)
12.0
4.1
27.2
5.2
59.1
50.7
12.0
3.4
4.3
—
—
9.8
4.8
9.8
15.5
26.1
28.0
13.7
13.2
12.3
21.1
—
42.0
35.0
25.5
24.8
30.1
27.8
15.6
8.1
11.6
5.9
13.9
35.4
22.1
26.1
4.6
4.8
3.7
4.3
8.9
13.2
4.1
14.5
50.8
83.3
191.5
24.4
120.5
86.6
108.5
104.2
105.2
50.3
46.2
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Movie S1. CAV-1 dynamics during ovulation in control animals. Live animals expressing GFP:CAV-1 were immobilized on 10% agarose pads in the presence of
0.1-μm diameter polystyrene microspheres. Time-lapse imaging was conducted by using swept-field confocal microscopy. Images were acquired at 20-s intervals.
Movie S1
Movie S2. CAV-1 dynamics during ovulation in fcho-1; ehs-1; itsn-1 triple mutant animals. Animals lacking all components of the FEI complex and expressing
GFP:CAV-1 were immobilized on 10% agarose pads in the presence of 0.1-μm diameter polystyrene microspheres. Time-lapse imaging was conducted by using
swept-field confocal microscopy. Images were acquired at 20-s intervals.
Movie S2
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Movie S3. MIG-14 dynamics during ovulation in control animals. Live animals expressing GFP:MIG-14 were immobilized on 10% agarose pads in the presence
of 0.1-μm diameter polystyrene microspheres. Time-lapse imaging was conducted by using swept-field confocal microscopy. Images were acquired at 20-s
intervals.
Movie S3
Movie S4. MIG-14 dynamics during ovulation in animals lacking all components of the FEI complex. Double mutant animals (fcho-1; itsn-1) expressing GFP:
MIG-14 were depleted of EHS-1 by using RNAi and immobilized on 10% agarose pads in the presence of 0.1-μm diameter polystyrene microspheres. Time-lapse
imaging was conducted by using swept-field confocal microscopy. Images were acquired at 20-s intervals.
Movie S4
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